The invention relates to the determination of the activity (concentration multiplied by reaction rate) of selected endopeptidases in body fluids by mass spectrometric measurements of the reaction products of added reporter substrates. Mass spectrometric diagnostics by analyses of substance mixtures extracted from body fluids are still in their infancy. This is true for both the development of diagnostic measurement and evaluation methods as well as for the official validation of the mass spectrometers or mass spectrometric procedures for diagnostic purposes. The first mass spectrometric methods which are validated for medical diagnostics are now coming onto the market. In Europe, the validation is based on an IVD Compatibility Declaration (CE) by manufacturers, who are subject to a special quality audit in accordance with DIN EN ISO 13485:2003, for example. IVD is the abbreviation for “in vitro diagnostics”. In Germany, this validation process is regulated by the Medical Products Act (MPG), which is based on the European Directive 98/79/EC. Outside Europe, validation directives usually are provided by official bodies.
In the publication “Differential exopeptidase activities confer tumor-specific serum peptidome patterns”, J. Villanueva et al., J. Clin. Invest., 116: 271-284 (2006), it was shown that peptides in blood serum which are produced by enzymatic digestion are not just useless garbage, as had largely been assumed, but that it is possible to find characteristic patterns therein to identify disease-specific enzyme activities. The authors were able to use the digest peptide patterns of the endogenous blood proteins which are present in higher concentrations, such as fibrinogen or other clotting factors, to distinguish between three different types of cancer as well as healthy control samples. The endogenous blood peptides including the digestion products of larger proteins were measured after a broadband extraction on magnetic beads with hydrophobic surfaces in a time-of-flight mass spectrometer with ionization by matrix-assisted laser desorption (MALDI), where all extracted peptides from a sample can be recorded simultaneously in a single mass spectrum. The analysis of these peptides showed that digestion reactions of the proteins brought about by the enzymes in the blood serum did not proceed in the same way in all samples but rather that the nature and rate of the reactions was different depending on the disease. Furthermore, it was possible to show that not all proteins in the blood are digested. Practically no digest peptides of the most prevalent proteins, i.e. the albumins and globulins, can be found. These highly molecular proteins are protected by their structure in such a way that they resist attacks by enzymes. The digest peptides could be mostly assigned to the clotting factors such as fibrinogen a or C3f.
Blood consists mainly of water (over 90%), various types of minute blood particles, small quantities of salts, several non-protein organic substances, and around seven percent is made up of proteins, of which albumins and globulins form the largest part. The next prevalent are the so-called clotting factors, above all fibrinogen. The peptidases which are of interest as possible biomarkers here are present in the blood samples at lower concentrations of usually far below 10−6 percent down to 10−10 percent, and elude a direct mass spectrometric measurement; they can therefore only be measured indirectly by their effects. One type of such an indirect measurement by an enzymatic effect has been described in the work cited above. Today, in a good mass spectrometer the direct measurement and evaluation of protein profiles from blood serum is generally limited to the concentration range of 10−1 to 10−4 percentage by weight.
The activity of the peptidases cannot only be measured in blood, but also quite generally in all body fluids. The term “body fluid” here shall therefore encompass all fluids of the body in the most general sense, i.e. in addition to blood also lymphs, liquor, cell homogenates and cell extracts, for example, and also fluids excreted by the body such as urine, sweat, lacrimal fluid and others, even if these analyses often focus on blood. If the term “blood sample” is used below, it can refer to “whole blood”, “blood serum” or also “blood plasma”. If the blood particles are removed from blood which has just been taken, the “whole blood”, by centrifuging, for example, the “blood plasma” is obtained, which still contains all clotting factors, above all fibrinogen. If it is to be stored or transported, the blood plasma must be prevented from coagulating by adding anticoagulants. If, on the other hand, the whole blood is coagulated, fibrinogen is broken down to fibrins through different stages with the assistance of other clotting factors. These fibrins polymerize and together with the blood corpuscles form the blood clot. If this blood clot is removed by centrifugation or any other means, one obtains the “blood serum”, which now contains (almost) no coagulants.
The smaller proteins with molecular weights of up to several thousand atomic mass units, which consist of only a few tens of amino acids, are called peptides; unless otherwise specifically mentioned, they are included here in the term “proteins”. The vast majority of peptides in body fluids are so-called “digest peptides” which are created as a result of the continuous enzymatic stronger or weaker digestion of larger proteins. In blood, the digestion concerns fibrinogen, for example, and in cells, endogenous proteins which are no longer needed. Proteins are digested by enzymes which are usually called “peptidases”, or also “proteases” or “proteinases” and which exist in hundreds or even thousands of different types in human and animal bodies. The peptidases are classified into endopeptidases and exopeptidases according to their type of effect.
“Endopeptidases” cleave proteins inside the amino acid chain of the proteins, but only if certain enzyme-specific “cleavage motifs” are present in the chain of the amino acids. One example of this is the familiar digest enzyme trypsin which always cleaves adjacent to the amino acids lysine and arginine. The cleavage motif at which a specific peptidase cuts can consist of a single specific amino acid and also of an enzyme-specific chain of several amino acids. Blood generally contains only endopeptidases, which have more complicated motifs of several amino acids and which are specialized in the digestion of certain proteins, because otherwise all blood proteins would be attacked in a life-threatening way.
“Exopeptidases”, on the other hand, indiscriminately break down peptides from the end: One amino acid after the other is removed, generally creating a mixture of digest peptides which each differ by one amino acid and thus enabling the sequence of the broken-down protein to be identified in a mass spectrometric measurement by virtue of the mass differences. Exopeptidases, which break down two or even three amino acids as a group, are less common. Depending on the exact type, exopeptidases work either from the C-terminal or from the N-terminal end of the protein (carboxyl exopeptidases and amino exopeptidases). The mixtures of digestion products created by exopeptidases are also called “digestion ladders”. The proteins naturally occurring in blood are usually protected by folding patterns which resist the attack of the ever-present exopeptidases on the terminal amino acids.
All enzymes have a catalytic effect on one or more target substances, which are termed the “substrate” of the enzyme, and which are modified by the catalytic activity of the enzyme in a way which is characteristic of the enzyme. The enzymes are therefore not used up by their activity, but rather their activity gradually decreases over quite long periods of some days, the activities of other enzymes or even self-digestion also playing a part. The half lives of the enzymes' activity amount to a few days; freezing prevents the activity from diminishing.
The rate of the catalytic reactions of the enzymes and hence the change to the substrates is very different. “Sluggish enzymes” have a reaction rate of around one substrate molecule per second and enzyme molecule; fast enzymes can exhibit a reaction rate of up to 100,000 substrate molecules per second and enzyme molecule. The fastest known enzyme is catalase, which breaks down hydrogen peroxide which is toxic to the body. Fast reaction rates require that sufficient substrate molecules are available, however, and also that diffusion does not restrict the supply. The peptidases, which digest proteins and peptides, usually have reaction rates of around 100 to 1,000 substrate molecules per second and per molecule if there is sufficient supply.
Before a mass spectrometric measurement, the reaction products of enzymes, for example, must be extracted from the body fluid. One option is to use broadband extractions, which bind almost all peptides from the sample to differently coated, actively binding solid surfaces, for example. Of greater interest here, however, are extraction methods which are selectively designed for different “anchor groups” and essentially only bind those molecules which are equipped with the anchor groups. This can occur via chelate-like bonded metal atoms of various types, via substance-specific ligand bonds, and also by custom-made protein-specific bonds similar to the antigen-antibody bonds, for example.
The indirect measurement of the enzyme activity in blood by measuring the reaction products is a breakthrough for diagnostic applications of biomarkers, but also has its disadvantages which are caused by the variability of the composition of blood. These disadvantages can largely be avoided by directing the enzyme activity towards artificial “reporter substrate molecules” added in a standardized way.
Endopeptidases, which have complicated cleavage motifs with a specific sequence of amino acids, are safely identified by the effect they have on appropriately composed reporter substrate molecules and their activity can be measured indirectly by the reaction products. Depending on the duration and rate of the reactions, the occurrence of the reaction products will be many orders of magnitude higher than the molecules of the endopeptidases themselves. If one molecule of an endopeptidase cleaves 100 molecules of a reporter substrate per second, for example, then one million cleavage product molecules per peptidase molecule are present after only three hours. This reaction rate is not even high, rather below average. The prerequisite, however, is the supply of a sufficiently large quantity of reporter substrate molecules, which must therefore be present in a very high concentration in order not to bring about any diffusion-controlled deceleration of the activity.
So if a certain endopeptidase is present in the blood at a concentration of only 10−8 percent then, after adding a suitable substrate at a concentration of around one percent, reaction products at a concentration of around one hundredth of a percent are present after only three hours incubation time. Around every hundredth substrate molecule is cleaved. This concentration is ideal for an extraction with subsequent mass spectrometric determination.
The “activity” of an enzyme is given by the product of its concentration and its reaction rate. The reaction rate of the reporter substrate molecules as a result of the endopeptidases is strongly dependent on the temperature, most enzymes operate best at 37° C. (approx. 99° F.), i.e. at human body temperature. A temperature which is around five to ten degrees Celsius (9-18° F.) lower reduces the rate of the reaction by around half each time. A reliable measurement of the activity therefore requires incubation under specified conditions. The pH value also plays a role and has to be controlled correctly.
The quantities of specific reaction products produced by the enzymes can serve as biomarkers to identify illnesses. It is known that different types of cancer secrete different types of endopeptidases to a much greater extent into the blood, said endopeptidases cleaving substrates with very specific cleavage motifs. The precise knowledge of their activity in the blood can be used to detect and identify these types of cancer.
DE 10 2006 009 083 A1 (J. Franzen et al.; corresponding to WO 2007/098859 A1) discloses a method of measuring the peptidase activity which uses the addition of exogenous probe substrates (called “reporter substrate molecules” here), which are each provided with an anchor group for a substance-specific extraction. Using exogenous substances with anchor groups as substrates to measure the peptidase activities eliminates many of the disadvantages which exist when analyzing digestion products of endogenous proteins. There is still the disadvantage that this method has a small dynamic range of measurement, however. In addition to the reaction products to be measured, the reporter substrate molecules which have not been broken down are also always extracted with the anchor group. For slow reactions or for very low peptidase concentrations, both of which supply only a small fraction of the reaction products in a given time, the measurement becomes extraordinarily difficult because of the presence of high concentrations of reporter substrate molecules which have not been broken down.